Restriction endonucleases are a class of enzymes that occur naturally in prokaryotic and eukaryotic organisms. When they are purified away from other contaminating cellular components, restriction endonucleases can be used in the laboratory to cleave DNA molecules into precise fragments. This property enables DNA molecules to be uniquely identified and to be fractionated into their constituent genes. Restriction endonucleases have proved to be indispensable tools in modern genetic research. They are the biochemical "scissors" by means of which genetic engineering and analysis are performed.
Restriction endonucleases act by recognizing and binding to particular sequences of nucleotides (the "recognition sequence") along the DNA molecule. Once bound, they cleave the molecule within, or to one side of, this sequence. Different restriction endonucleases have affinity for different recognition sequences. About 100 kinds of different endonucleases have so far been isolated from many microorganisms, each being identified by the specific base sequence it recognizes and by the cleavage pattern it exhibits. In addition, a number of restriction endonucleases, called restriction endonuclease isoschizomers, have been isolated from different microorganisms which in fact recognize the same recognition sequence as those restriction endonucleases that have previously been identified. These isoschizomers, however, may or may not cleave the same phosphodiester bond as the previously identified endonuclease.
In nature, restriction endonucleases play a protective role in the welfare of the microbial cell. They enable the microorganism to resist infection by foreign DNA molecules like viruses and plasmids that would otherwise destroy or parasitize them. They achieve this resistance by scanning the lengths of the infecting DNA molecule and cleaving them each time that the recognition sequence occurs. The DNA cleavage that takes place disables many of the infecting genes and renders the DNA susceptible to further degradation by non-specific exonucleases.
A second component of microbial protective systems are the modification methylases. Modification methylases are complementary to their corresponding restriction endonucleases in that they recognize and bind to the same recognition sequence. Modification methylases, in contrast to restriction endonucleases, chemically modify certain nucleotides within the recognition sequence by addition of a methyl group. Following this methylation, the recognition sequence is no longer bound or cleaved by the restriction endonuclease. The microbial cell modifies its DNA by virtue of its modification methylases and therefore is completely insensitive to the presence of its endogenous restriction endonucleases. Thus, endogenous restriction endonuclease and modification methylase provide the means by which a microorganism is able to identify and protect its own DNA, while destroying unmodified foreign DNA.
The combined activities of the restriction endonuclease and the modification methylase are referred to as the restriction-modification system. Three types of restriction-modification systems have been identified that differ according to their subunit structure, substrate requirements and DNA cleavage. Specifically, Type-I and Type-III restriction systems carry both modification and ATP-requiring restriction (cleavage) activity in the same protein. Type-II restriction-modification systems, on the other hand, consist of a separate restriction endonuclease and modification methylase, i.e., the two activities are associated with independent proteins.
Type II restriction endonucleases are endodeoxyribonucleases which are commonly used in modern genetic research. These enzymes recognize and bind to particular DNA sequences and once bound, cleave within or near this recognition sequence. Phosphodiester bonds are thereby hydrolyzed in the double stranded DNA target sequence, i.e., one in each polynucleotide strand. Type-II restriction endonucleases can generate staggered breaks within or near the DNA recognition sequence to produce fragments of DNA with 5' protruding termini, or DNA fragments with 3' protruding termini. Other Type-II restriction endonucleases which cleave at the axis of symmetry, produce blunt ended DNA fragments. Therefore, Type-II restriction endonucleases can differ according to their recognition sequence and/or the location of cleavage within that recognition sequence.
Type-II restriction endonucleases are frequently used by the genetic engineers to manipulate DNA in order to create novel recombinant molecules. Specific Type-II restriction endonucleases are known for numerous DNA sequences, but there is still a need to provide improved means for producing Type-II restriction endonucleases. Therefore, it is an object of the present invention to make commercial production of these enzymes more practical by using recombinant DNA technology.
There has been much effort to clone type II restriction-modification systems. The first cloning of a DNA endonuclease gene was described by Mann MB et al., Gene 3:97-112 (1978). Since then more than seventy DNA methylase and restriction endonucleases have been cloned, the majority of the restriction endonuclease genes being closely linked to its corresponding methylase gene. Cloning of such genes allows one to produce large quantities of an enzyme.
Several methods by which restriction-modification systems can be cloned have been described. A number of endonuclease and methylase genes have been cloned from endogenous plasmids: EcoRII (Kosykh VB et al. (1980) Mol. Gen. Genet. 178:717-718), EcoRI (Newman AK et al. J. Biol. Chem. 256:2131-2139 (1981), and Greene PJ et al., J. Biol. Chem. 256:2143-2153 (1981)), EcoRV (Bougueleret L et al. Nucl. Acids Res. 12:3659-3676 (1984)), PvuII (Blumenthal RM et al. J. Bacteriol. 164:501-509 (1985)), and PaeR71 (Gingeras TR and Brooks JE Proc. Natl. Acad. Sci. USA 80:402-406 (1983)). Other methods of cloning include a phage restriction method in which bacterial cells carrying cloned restriction and modification genes will survive phage infection (Mann et al. supra; Walder RY et al. Proc. Natl. Acad. Sci. U.S.A. 78:1503-1507 (1981); and Rodicio MR and Chater KF Mol. Gen. Genet. 213:346-353 (1988)), and a procedure based on methylation protection suggested by Mann et al., supra, and Szomolanyi E et al. Gene 10:219-225 (1980). This latter scheme involves digestion of a plasmid library with the restriction enzyme to be cloned so that only plasmids whose sequences are modified, because of the presence of the methylase, will produce transformants in a suitable host. This selection has worked well to clone endonuclease and methylase genes together as well as methylase genes alone (Szomolanyi et al., supra; Janulaitis A et al. Gene 20:197-204 (1982); Walder RY et al. J. Biol. Chem. 258:1235-1241 (1983); Kiss A and Baldanf F Gene 21:111-119 (1983); and Wilson GG Gene 74:281-289 (1988)). However, this technique sometimes yields only the methylase gene, even though the endonuclease and modifying genes are closely linked.
Cloning of certain restriction-modification systems in E. coli, including DdeI (Howard KA et al. Nucl. Acids Res. 14:7939-7950 (1989)), BamHI (Brooks JE et al. Nucl. Acids Res. 17:979-997 (1989)), and KpnI (disclosed herein), has required a multi-step approach. In each case, protection of the host with methylase expressed on a plasmid was necessary to stabilize a compatible vector containing the functional endonuclease gene. A head-start model to explain why some restriction-modification systems must be cloned utilizing a protected host was proposed by Wilson; supra. This model states that in order to establish a plasmid carrying a restriction-modification system, methylase protection must be faster than endonuclease digestion. Otherwise, restriction enzyme would cleave unmethylated plasmid and/or genomic DNA thereby killing the host. Although this model is a plausible explanation of plasmid establishment, it has not been determined previously whether continued independent expression of methylase from a separate plasmid is necessary to maintain the plasmid carrying the restriction-modification system during cell growth and replication.